线粒体自噬-Warburg效应介导apelin促血管平滑肌细胞增殖

Applicant first reported NADPH oxidase 4 (NOX4)-derived reactive oxygen species (ROS) mediated APJ receptor endogenous ligand apelin-13 stimulate vascular smooth muscle cells (VSMC) proliferation; Preliminary experiments found that apelin-13 increases VSMC AMPKα phosphorylation, induces mitophagy， promote lactate production under aerobic conditions(aerobic glycolysis reaction that Warburg effect), pursuant to that "mitophagy-Warburg effect pathway mediated vascular smooth muscle cell proliferation induced by apelin" scientific hypothesis. Topics to be adopted RNA interference, to explore whether apelin-13-induced VSMC mitophagy; Clear whether apelin-13 induced VSMC Warburg effect; analyze whether mitophagy regulate apelin-induced VSMC Warburg effect; Reveal mitophagy-Warburg effect mediated apelin-13 induced VSMC proliferation. From mitophagy-Warburg effect new perspective ,to clarify apelin / APJ system to promote VSMC proliferation of new mechanisms.To provide a basis for revealing the pathogenesis of atherosclerosis and other vascular disorders.

申请者首先报道NADPH氧化酶4（NOX4）源性活性氧（ROS）介导APJ受体内源性配体apelin-13促血管平滑肌细胞（VSMC）增殖；预实验发现apelin-13 上调VSMC的AMPKα磷酸化、诱导线粒体自噬、促进有氧条件下乳酸生成（有氧糖酵解反应即Warburg效应），据此提出"线粒体自噬-Warburg效应介导apelin促血管平滑肌细胞增殖"科学假说。课题拟采用RNA干扰等方法，探讨apelin-13是否诱导VSMC线粒体自噬；明确apelin-13是否诱导VSMC Warburg效应；分析线粒体自噬是否调节apelin-13诱导的VSMC Warburg效应；揭示线粒体自噬-Warburg效应是否介导apelin-13促VSMC增殖。课题从线粒体自噬-Warburg效应新视角阐明apelin/APJ系统促VSMC增殖新机制，为揭示动脉硬化等血管增生性疾病发病机理提供依据。

课题从线粒体自噬-Warburg效应新视角阐明apelin/APJ系统促VSMC增殖新机制，为揭示AS等血管增生性疾病发病机理提供依据。研究发现：1. Apelin-13浓度和时间依赖性促进VSMC内PINK1、parkin 、p-parkin、VDAC1、LC3II/I表达；2. Apelin-13浓度和时间依赖性下调p62表达，促进 TOM20、Mfn1、Mfn2、OPA1、Drp1表达；3. Apelin-13浓度及时间依赖性促VSMCs 的p-AMPKα表达；4. siRNA-AMPKα减弱apelin-13对VSMCs 的Beclin1，OPA1，Mfn2，Mfn1及Tom20的影响，确证了apelin-13通过AMPKα途径诱导VSMC线粒体自噬；5. 在有氧条件下，apelin-13能够浓度依赖性促进VSMC乳酸生成，促进PKM2、LDHA、MCT4、GLUT1表达；6. F13A阻断APJ受体或沉默APJ受体，apelin-13的作用减弱，沉默PKM2、LDHA，apelin-13引起的VSMC乳酸生成减少；7.干扰PINK1、parkin表达减弱apelin-13对VSMCs 的葡萄糖摄取、乳酸生成和转运、ATP生成、PKM2、LDHA及MCT1、MCT4表达；8. Apelin-13浓度和时间依赖性促进VSMC的PCNA表达，促进AS发生发展；9. 敲除PINK1后，aplein-13诱导的VSMC线粒体自噬及细胞增殖明显减弱，parkin 、p-parkin、VDAC1、LC3II/I表达下调，apelin-13加剧apoE敲除小鼠主动脉壁AS发展。10. 缺氧、饥饿、雷帕霉素、高尔基体应激诱导剂、Apelin-13、过表达APJ、Ang II、压应力诱导VSMCs内的TGN46、GM130和LC3荧光共定位及高尔基体自噬；11. Apelin-13/APJ上调GOLPH3的表达，干扰GOLPH3的表达抑制高尔基体自噬。以上结果基本阐明线粒体自噬-Warburg效应介导apelin-13促VSMCs增殖机理，发现apelin/APJ系统诱导VSMCs线粒体自噬和Warburg效应的新生物学功能，为揭示AS等增生性心血管疾病发生发展机理提供新的依据。课题组首次报道高尔基体自噬的存在，为揭示血管平滑肌增生性心血管疾病提供了病理生理基础和理论依据。